One of the most significant recent realizations in biology and medicine is the key role that noncoding RNAs (ncRNAs) play in numerous and important biological processes including many aspects of gene expression. Not surprisingly, the impact of ncRNA function on human health and disease is proving to be broad and substantial. Most non-coding RNAs assemble with partner proteins to form non-coding RNA-protein complexes (ncRNPs), and these function in diverse cellular processes. The major objective of this proposal is to obtain a detailed understanding of the biogenesis and function of two distinct and important classes of ncRNPs through biochemical and molecular analysis. Our first aim is to determine how the four essential proteins and guide RNA of the H/ACA RNP assemble and function together to carry out RNA modification. In humans, H/ACA RNPs are required for the production of the pre-mRNA splicing and protein translation machinery as well as for genome stability. Our work on this complex has provided new insight into the etiology of dyskerotosis congenita. The experiments proposed in our second aim will provide substantial information on the biogenesis and function of a newly recognized prokaryotic genome defense system. This pathway is strongly implicated in defense against viruses and other mobile genetic elements in prokaryotes including important human pathogens. This system represents a major new RNA silencing pathway with the potential to provide insight on other silencing pathways. Importantly, this pathway may provide a means for both experimental gene silencing and development of novel classes of therapeutics effective in the broad range of prokaryotes in which the system is found. Together, the proposed studies have great potential for significant contributions to human health and disease.